Mitogen-activated protein kinase (MARK) pathways are evolutionary-conserved eukaryotic signaling modules that regulate diverse cellular processes. In response to a multitude of extracellular stimuli, MAPKs phosphorylate and activate downstream transcription factors that modify chromatin and orchestrate gene expression. Many MARK pathways have been genetically defined in Saccharomyces cerevisiae. In this proposal we focus on one of the well-characterized yeast MARK pathways, the high osmolarity glycerol (HOG) response pathway. During the last few decades, the components and the regulatory network of this cascade have been elucidated using genetic and biochemical assays performed on large populations of cells. In this proposal we take a complementary approach by monitoring the signaling dynamics in single cells with high temporal resolution. This method provides three distinct advantages compared to traditional biochemical and genetic assays on cell populations: (1) MARK activity will be measured in live cells;(2) measurements are almost instantaneous giving a temporal resolution on the order of 1 second which is the typical time-scale of the kinetics of individual reaction steps;(3) measurements will be performed on many single cells in parallel, allowing studies of cell-to-cell variability in signaling dynamics and allowing a direct measurement of the correlation between the concentration of a key component in the network and the signaling dynamics in a single cell. The specific aims are organized around measuring the signaling dynamics at three different time-scales: (1) the initial rapid signal propagation before feedback regulation is activated;(2) adaptation dynamics dominated by feedback regulation and (3) evolutionary time-scales, which will be explored by using a comparative experimental analysis of the HOG signaling pathway of related yeast species. The proposed experimental work will be closely integrated with quantitative modeling approaches with the ultimate goal to build a predictive model of the HOG response pathway. Relevance to public health: Some mammalian MAPKs are activated by inflammatory cytokines and environmental stresses which might play an important role in diseases like asthma and autoimmunity. Because MARK cascades are highly conserved, the proposed experimental and theoretical techniques and concepts that will be important for the analysis of MARK cascades in higher eukaryotes.